Chemical synthesis of RNA is a more difficult task than chemical synthesis of DNA, because the 2′-hydroxyl group in the ribose has to be protected during chemical synthesis. The close proximity of a protected 2′-hydroxyl to the internucleotide phosphate may present problems, both in terms of formation of the internucleotide linkage and in the removal of the 2′-protecting group once the oligoribonucleotide is synthesized. In addition, the internucleotide bond in RNA is less stable than that in DNA.
Until recently, the typical approach to RNA synthesis utilized ribonucleotide monomers in which the 5′-hydroxyl group was protected by the acid-labile dimethoxytrityl (DMT) protecting group, which can be removed under acidic conditions after coupling of the monomer to the growing oligoribonucleotide. Various acid-stable protecting groups have been placed on the 2′-hydroxyl to prevent isomerization and cleavage of the internucleotide bond during the acid deprotection step, for example the tert-butyldimethylsilyl group, known as TBDMS (Ogilvie et al., 1979). The use of TBDMS as 2′-protecting group dominated the previously small market for RNA chemical synthesis for a very long time (Usman et al., 1987; Ogilvie et al., 1988).
However, oligoribonucleotide syntheses carried out using TBDMS are by no means satisfactory and may produce RNA products of poor quality. In some cases the coupling efficiency of these monomers is decreased due to steric hindrance of the 2′-TBDMS protecting group, which may affect the yield and purity of the full-length product, and also limit the length of the oligoribonucleotide that can be achieved by this method. Furthermore, in some cases, the synthesis of the monomer (e.g., 5′-O-DMT-2′-O-TBDMS-ribo-3′-O-(beta-cyanoethyl-N,N-diisopropyl)phosphoramidite) can be both challenging and costly due to the non regiospecific introduction of the TBDMS group on the 2′-hydroxyl and to the migration of the silyl group from the 2′ to the 3′ position, that occurs during subsequent steps of the synthesis of the monomer.
The demand for synthetic RNA has increased in the past decade, largely due to the discovery of RNA interference. To meet this growing need, it is desirable to develop improved RNA synthesis schemes, particularly 2′-protecting groups that can be introduced at low cost in high yield, along with stream-lined deprotection methods.